The broad aim of our studies in this project continues to be the elucidation of the role of different plaque organisms as caries-etiologic agents and the integration of various putative cariogenic determinants into a unified concept of caries causation. A major effort will involve the design and validation of 16S rRNA-based DNA probes for plaque organisms. Target organisms will be S. mutans, S. sobrinus and species comprising the non-mutans streptococci (non-MS) (Species-specific probes), Actinomyces, Bifidobacterium, Lactobacillus and Veillonella (genous-specific probes for all), and perhaps some other bacteria. In contrast to other available methods, the probes will permit the rapid and accurate enumeration of these caries-relevant plaque organisms. Their association with human root caries will be determined in a large clinical study (Clinical Core). Of particular interest in this regard will be to so-called 'low'-pH non-MS i.e., a segment of the non-MS capable of acidogeneis at low pH as indicated by their 'final' pH in sugar broth. Our previous findings have suggested that, besides the mutans streptococci, these organisms warrant serious consideration as a, perhaps, second echelon of cariogenic plaque organisms. Further, DNA probe analysis may help to unravel further the difficult taxonomy of these and other oral streptococci. Probe identification of these non-MS species will be coupled with analysis of their 'final' pH. Homogeneity of the 'final' pH of strains of the same probe-identified species will permit direct quantitation of the 'low-pH' non-MS segment in plaque. Data on 'final' pH will also be obtained for the other target organisms using samples from the large study (Clinical Core) and other sources (e.g. Culture Collection). Similar kind of data will be obtained for these organisms with respect to their acid tolerance (ability to grow/survive at acidic pH). This will yield a comprehensive picture of the plaque fora with respect to two major caries-relevant bacterial traits, i.e. capability for acidogenesis at acid plaque pH and acid tolerance, and will further aid in the construction of an integrated concept of caries causation. Studies of the in situ identification of bacteria with immunocytochemistry in relation to root caries will focus initially on a comprehensive identification of organisms in the 'advancing bacterial front' of dentin. This will be coupled with an analysis of the mineral content of areas of altered dentin which, according to our findings, appear to be due to the action of single types of organisms. This work may yield highly-significant new data on the cariogenicity of different plaque organisms. Later, this work will be expanded with a comparison of the composition of the plaque flora associated with intact and altered cementum or dentin.